The activation of dioxygen in biological systems is a question of great importance because of its involvement in many metabolic processes. Although the mechanism of oxygen activation in heme systems such as cytochrome P450 and peroxidases is becoming quite well understood, much less is known of the corresponding mechanisms for non-heme systems. These latter enzymes are involved in aromatic and aliphatic metabolism, collagen formation, the synthesis of neurochemicals and antibiotics, and participate in reactions that range from oxidative cleavage of aromatic rings, aliphatic and aromatic hydroxylation, to ring cyclizations involving heteroatoms. For many mononuclear nonheme iron enzymes, the reaction with O2 occurs only after substrate or cofactor interacts with the active site iron, e.g. catechol for catechol dioxygenases, substrate thiol for isopenicillin N synthase, and the alpha-ketoacid cofactor for prolyl hydroxylase, clavaminate synthase and other alpha-ketoglutarate-dependent oxygenases. It is proposed that a common general mechanism unites the chemistry of these enzymes, but the details of the dioxygen activation step vary from enzyme because of the divergent natures of the substrates and/or cofactors. Important questions include, 1) how is O2 activated by these enzymes, and 2) what is the nature of high valent intermediates that are involved in the reactions? Spectroscopic (NMR, EPR, resonance Raman, Mossbauer, EXAFS) and mechanistic studies are proposed for a number of enzymes to elucidate details of the active site structure and the mechanism of action. These include intradiol cleaving catechol dioxygenases, isopenicillin N synthase, p- hydroxyphenylpyruvate dioxygenase, and clavaminate synthase (ketoacid- dependent enzymes). Model complexes will be synthesized and used as models to interpret spectroscopic features of the enzymes. More importantly, the key mechanistic steps of the oxidations will be modeled. Areas of particular interest are: 1) biomimetic oxidative cleavage of catechols, 2) development of models for the isopenicillin N synthase and the alpha- ketoacid-dependent oxygenase reactions, 3) characterization of transient high valent intermediates. As in the past, the synergistic interaction of the biochemical and inorganic aspects of the proposal is important for the success of the program.